Spiral and contraction-expansion coupled microchannel and flexible microalgae sorting method

Abstract

The invention discloses a spiral and contraction-expansion coupled micro-channel, which is formed by coupling a spiral micro-channel and a contraction-expansion channel according to three different forms of single-side coupling, double-side coupling or staggered coupling, and is provided with a sample inlet and two outlets, the initial radius of the spiral and contraction and expansion coupling micro-channel is 3-5 mm. The invention further discloses a flexible microalgae sorting method based on the spiral and contraction-expansion coupled microchannel. According to the spiral and contraction-expansion coupled micro-channel provided by the invention, the problems that a traditional microalgae cell sorting method is greatly influenced by environmental conditions and growth states, the sorting speed is low and professional experimental equipment is needed are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of flexible microalgae separation, relates to a spiral and contraction-expansion coupling microchannel, and also relates to a method for flexible microalgae sorting by using the spiral and contraction-expansion coupling microchannel. Background Art

[0002] In freshwater systems, Chlorella and Haematococcus pluvialis are two algae with great research value. Chlorella is the earliest developed algae protein and one of the most productive species in the world's microalgae industry. Haematococcus pluvialis is recognized as the most ideal source of natural astaxanthin in nature. Astaxanthin is the most efficient pure natural antioxidant, showing good biological activity in scavenging free radicals, anti-aging, anti-tumor and immune regulation. The separation of Chlorella rich in algae protein and Haematococcus pluvialis rich in astaxanthin is an important part of microalgae biology and food chemistry research.

[0003] Traditional microalgae cell sorting techniques mainly include micropipette sorting, spread plate differential centrifugation and flow cytometry. Chlorella and Haematococcus pluvialis will compete for nutrients in freshwater systems, and the two microalgae cells tend to produce similar green colonies, making it difficult to distinguish the colonies. In order to better study a single algae, the two algae need to be sorted. However, traditional microalgae cell sorting methods have some defects, such as being affected by environmental conditions and growth conditions, small differences between some similar species, slow sorting speed, and requiring professional operating skills and expensive experimental equipment.

[0004] Several high-throughput, high-precision, and low-cost microfluidic separation methods have been developed for microalgae. Some techniques utilize external physical fields, such as dielectrophoresis, magnetoelectrophoresis, and acoustophoresis for microalgae separation. However, external physical fields may provide additional stimulation to the cells, and these methods usually require complex manufacturing processes and bulky experimental setups. Other microalgae separation techniques use inherent channel structures, such as inertial focusing and elastic inertial focusing. Compared with elastic focusing, inertial microfluidics does not require complex viscoelastic sample configuration, does not require any external force field, and relies on the designed channel structure for sorting.

[0005] Particle size is the main parameter for inertial focusing and sorting, and the critical threshold of particle separation depends on the blockage ratio, which is the ratio of particle size to the hydraulic diameter of the channel. Our previous work reported that large particles can only be separated from small particles when the blockage ratio of the spiral channel exceeds a certain value. For the separation of microalgae cells, the cell size is predetermined, and the design principle of the coupled channel still needs to consider the blockage ratio. However, in existing microfluidic devices, spiral channels and contraction-expansion channels are mostly spliced ​​or multiple repetitions of a single channel. Only the design criteria of the two different channels need to be considered separately. At present, there is no integrated thinking to couple the two to design channel parameters and use them for microalgae sorting. Summary of the invention

[0006] The purpose of the present invention is to provide a spiral and contraction-expansion coupling microchannel, which solves the problems that the traditional microalgae cell sorting method is greatly affected by environmental conditions and growth status, has a slow sorting speed and requires professional experimental equipment.

[0007] Another object of the present invention is to provide a method for flexible microalgae sorting using the spiral and contraction-expansion coupling microchannel.

[0008] The first technical solution adopted by the present invention is that the spiral and contraction-expansion coupling microchannel is composed of a spiral microchannel and a contraction-expansion channel coupled in three different forms of single-side coupling, double-side coupling or staggered coupling, the spiral and contraction-expansion coupling microchannel has a sample inlet and two outlets, and the initial radius of the spiral and contraction-expansion coupling microchannel is 3-5 mm; The cross section of the spiral microchannel is rectangular. The contraction cavity and the expansion cavity in the spiral and contraction-expansion coupling microchannel are set at an equal angle of 5°. The cross-sectional size of the contraction cavity is 50ⅹ50µm, and the cross-sectional size of the expansion cavity is 200ⅹ50µm.

[0009] The second technical solution adopted by the present invention is a method for flexible microalgae sorting based on a spiral and contraction-expansion coupled microchannel, using the above-mentioned spiral and contraction-expansion coupled microchannel, and the specific steps are as follows: Step 1, preparing a fluorescent polystyrene microsphere particle suspension; Step 2, preparing a microalgae cell mixture; Step 3, conduct velocity distribution simulation experiments on three types of microchannels to obtain simulation results; Step 4: Based on the simulation results, a fluorescent polystyrene microsphere particle suspension is used to conduct preliminary experiments on the inertial migration behavior of particles in three different microchannels to obtain preliminary experimental results; Step 5. Based on the preliminary experimental results, three different spiral and contraction-expansion coupled microchannels were used to analyze and compare the inertial separation behaviors of Chlorella and Haematococcus pluvialis, and the performance of three different microchannel chips for flexible microalgae sorting was evaluated to obtain the optimal separation parameters and complete the sorting of flexible microalgae.

[0010] The present invention is also characterized in that: The specific method of step 1 is: take fluorescent polystyrene microspheres with diameters of 5 μm and 15 μm respectively, add deionized water to dilute, and prepare two single-sized particle suspensions, wherein the volume ratio of each fluorescent polystyrene microsphere to deionized water is 1:24, and then mix the two single-sized particle suspensions at a volume ratio of 1:1, add Tween 20 solution to the mixed solution, the volume ratio of Tween 20 solution to the mixed solution is 1:200, oscillate evenly, and finally obtain a fluorescent polystyrene microsphere particle suspension by ultrasonic treatment.

[0011] The specific process of step 2 is: Step 2.1, cultivation of microalgae and collection of microalgae mixture: Cultivate common Chlorella and Haematococcus pluvialis in BG11 medium respectively, place in conical flasks, grow the cultures in a fully automatic intelligent light incubator, and receive a 12 / 12 hour light and dark cycle, and control the relevant temperature at 23-25°C. Shake the cultures continuously every day to ensure uniform nutrients and dispersion of cells; A phosphate buffer solution with a pH of 7.2-7.4 was taken and dripped into deionized water to prepare a PBS solution for later use, and the volume ratio of deionized water to phosphate buffer solution was 10:1; Step 2.2, process the microalgae samples: take the cultured Haematococcus pluvialis and Chlorella, respectively, and coarsely filter the Haematococcus pluvialis and Chlorella with 20 µm and 10 µm filters, filter out the cell precipitate, and then filter out the oversized cells again with 18 µm and 6 µm precision filters, respectively. Then, put them into a high-speed desktop centrifuge at a speed of 6000-6500 rpm and a temperature of 20-22°C for 4-5 min, pour out the supernatant, take out the algae cells, add the prepared PBS solution to obtain two algae cell solutions, the PBS concentration range of each algae cell solution is 0.1-0.5M, finally, take out the same volume of Haematococcus pluvialis solution and Chlorella solution, mix them, add sorbitol solution to the mixed solution and shake it thoroughly to obtain a microalgae cell mixed solution, and the volume ratio of the mixed solution to the sorbitol solution is 200:1.

[0012] The specific process of step 4 is: The microfluidic device was mounted on a bracket, and two fluorescent polystyrene microsphere particle suspensions were injected into the channel using a syringe pump, wherein the flow rate during injection into the channel was 50 µL / min, 100 µL / min, 150 µL / min, 200 µL / min, 300 µL / min, or 400 µL / min, and a constant flow rate of 10-400 μL / min was maintained. The entire device was then placed on the stage of an inverted microscope, and a high-speed camera was used to monitor and record the flow of the sample in the microchannel; 5 μm fluorescent polystyrene microspheres were used to simulate the behavior of Chlorella in the microchannel, and the focusing effects of the particles were compared at flow rates of 50 μL / min, 100 μL / min, 150 μL / min, 200 μL / min, 300 μL / min, and 400 μL / min; 15 µm fluorescent polystyrene particles were used to simulate the behavior of Haematococcus pluvialis in the microchannel. The flow rate of 15 µm fluorescent polystyrene particles was increased and the flow rate was controlled at 100-400 µL / min. The focusing effect of the particles was compared at flow rates of 100 µL / min, 200 µL / min, 300 µL / min and 400 µL / min, with a gradient of 100. By analyzing the focusing conditions of the two fluorescent polystyrene particles, the optimal flow rate for sorting the two mixed particles was 100-200 µL / min.

[0013] In step 5, a syringe pump is used to introduce the two types of microalgae cells into three different microchannels, respectively, wherein the flow rate during injection into the channel is 100-400 µL / min.

[0014] The specific method of using the spiral contraction and expansion single-side coupling microchannel chip for flexible microalgae sorting in step 5 is: The separation of Chlorella vulgaris and Haematococcus pluvialis was analyzed using a spiral contraction-expansion single-sided coupling flow channel. By analyzing the focusing effects of the two cells at different flow rates, it was found that the flow rate with the best separation effect was 200 µL / min.

[0015] The specific method of using the spiral contraction and expansion double-sided coupling microchannel chip for flexible microalgae sorting in step 5 is as follows: The separation of Chlorella vulgaris and Haematococcus pluvialis was analyzed using a spiral contraction-expansion double-sided coupling flow channel. By analyzing the focusing effects of the two cells at different flow rates, it was found that the flow rate with the best separation effect was 200 µL / min.

[0016] The specific method of using the spiral contraction and expansion staggered coupling microchannel chip for flexible microalgae sorting in step 5 is as follows: The separation of Chlorella vulgaris and Haematococcus pluvialis was analyzed using a spiral contraction-expansion staggered coupling flow channel. By analyzing the focusing effects of the two cells at different flow rates, it was found that the flow rate with the best separation effect was 200 µL / min.

[0017] When evaluating the performance of flexible microalgae sorting using three different microchannel chips in step 5, in addition to visual confirmation using a high-speed camera, the indicators can also be evaluated in terms of cell purity, recultivability, etc.; Among them, the specific formula for calculating purity EP is: EP = Na (outlet i ) / N total (outlet i ) (1) In the formula, N a (outlet i ) is the number of target cell population a in outlet i, N total (outlet i ) is the total number of cells extracted from this outlet.

[0018] The beneficial effects of the present invention are: (1) The present invention provides three new ways to couple spiral channels with contraction-expansion channels in microfluidic devices. These methods can be completed in a normal environment and growth state without the use of complex professional equipment. This solves the problems that traditional microalgae cell sorting methods are greatly affected by environmental conditions and growth states, have slow sorting speeds, and require professional experimental equipment. The three different coupling methods of spiral channels with contraction-expansion channels provide new ideas for the innovation of microchannels and new possibilities for the optimization of channel structure design. (2) The present invention provides microchannel separation based on three different coupling modes of microalgae cell size. The spiral microchannel introduces three different periodic expansion structures in the channel. The device can generate vortices and exert vortex-induced lift on the particles. The interaction between this force and the Dean drag force enables small particles to migrate stably without being restricted by the channel structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the spiral contraction and expansion single-side coupling microchannel structure of the present invention; Figure 2 Schematic diagram of the spiral contraction and expansion double-side coupling microchannel structure of the present invention; Figure 3 It is a schematic diagram of the spiral contraction and expansion staggered coupling microchannel structure of the present invention; Figure 4 It is a velocity flow field diagram of the first ring of the spiral contraction-expansion single-side coupling flow channel of the present invention; Figure 5 It is the velocity distribution diagram of the spiral shrinkage and expansion single-side coupling sorting chip of the present invention; Figure 6 It is a pressure profile diagram from the inlet to the outlet of the spiral shrinkage and expansion single-side coupling sorting chip of the present invention; Figure 7It is a graph of focused particles and numerical analysis results of the spiral contraction and expansion single-side coupling sorting microchannel of the present invention; Figure 8 The particle trajectory and the particle trajectory grayscale image of the 5 μm fluorescent polystyrene microsphere at the outlet of the spiral contraction and expansion single-side coupling flow channel of the present invention; Fig. 9 The particle trajectory and the particle trajectory grayscale image of the 15 μm fluorescent polystyrene microsphere at the outlet of the spiral contraction and expansion single-side coupling flow channel of the present invention; Fig.10 It is the separation effect and particle trajectory grayscale image of mixed microalgae in the spiral contraction and expansion single-side coupling flow channel at a flow rate of 200 μL / min of the present invention; Fig.11 It is a schematic diagram of the purity of microalgae cells in the collected liquid at each outlet in the spiral contraction-expansion single-side coupling flow channel of the present invention at a flow rate of 200 μL / min; Fig.12 This is a graph showing the 7-day cultivation results of the mixed microalgae of the present invention at two outlets of algae in a spiral contraction-expansion single-side coupling flow channel; Fig.13 This is the separation effect of the mixed microalgae in the spiral contraction and expansion double-side coupling flow channel at 150 µL / min and 200 µL / min of the present invention; Fig.14 This is the separation result of the mixed microalgae at the outlet of the spiral contraction-expansion double-side coupling flow channel at 150 µL / min and 200 µL / min of the present invention; Fig.15 It is a schematic diagram of the purity of microalgae cells in the liquid collected at each outlet in the spiral contraction-expansion double-sided coupling flow channel of the present invention at a flow rate of 200 μL / min; Fig.16 This is a graph showing the 7-day cultivation results of the mixed microalgae of the present invention at two outlets of algae in the spiral contraction-expansion double-sided coupling flow channel; Fig.17 It is the separation effect of the mixed microalgae of the present invention in the spiral contraction-expansion staggered coupling flow channel at 150 µL / min and 200 µL / min; Fig.18 This is the separation result of the mixed microalgae at the outlet of the spiral contraction-expansion double-side coupling flow channel at 150 µL / min and 200 µL / min of the present invention; Fig.19 It is a schematic diagram of the purity of microalgae cells in the collected liquid at each outlet in the spiral contraction-expansion staggered coupling flow channel of the present invention at a flow rate of 200 μL / min; Fig. 20 This is a graph showing the 7-day cultivation results of the mixed microalgae of the present invention at two outlets of the algae in the spiral contraction-expansion staggered coupling flow channel. DETAILED DESCRIPTION

[0020] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] The spiral and contraction-expansion coupled microchannel is composed of a spiral microchannel and a contraction-expansion channel coupled in three different forms: single-side coupling, double-side coupling or staggered coupling. The obtained microchannels are a spiral contraction-expansion single-side coupled microchannel, a spiral contraction-expansion double-side coupled microchannel and a spiral contraction-expansion staggered coupled microchannel, respectively. Figures 1 to 3 As shown, the spiral and contraction-expansion coupling microchannel has a sample inlet and two outlets, and the initial radius of the spiral and contraction-expansion coupling microchannel is 3~5 mm to prevent chip breakdown.

[0022] The cross section of the spiral microchannel is rectangular. For the selection of the flow channel width, the design criteria of the contraction-expansion channel need to be considered. The contraction chamber and the expansion chamber in the spiral and contraction-expansion coupling microchannel are set at an equal angle of 5°. The adjacent contraction chamber and expansion chamber are approximately 1:1 arc. Due to the characteristics of the Archimedean spiral, with the increase and diffusion of the number of turns, the arc of the contraction chamber and the expansion chamber will increase under the equal angle design. The width of the contraction chamber is 50 µm, and the width of the expansion chamber is 200 µm. In order to ensure the processing accuracy, the channel height is selected as 50 µm, thereby determining the cross-sectional dimensions of the contraction chamber to be 50ⅹ50µm, and the cross-sectional dimensions of the expansion chamber to be 200ⅹ50µm; In the particle focusing of the three coupled chip flow channels, if the flow channel length is insufficient, the particles will not have time to migrate to the equilibrium position; if the flow channel length is too long, the chip flow resistance will increase. Therefore, it is necessary to optimize the flow channel length reasonably. Due to the coupling of the contraction-expansion channel, the unnecessary flow channel length will be shortened through reasonable optimization while ensuring the sorting effect, so as to reduce the overall flow resistance of the chip. In this application, 72 sets of contraction-expansion channels are introduced, which are shortened from the previous 5 circles to 2 circles, and the length is greatly reduced.

[0023] Example 1 The method for flexible microalgae sorting based on spiral and contraction-expansion coupled microchannel uses the spiral and contraction-expansion coupled microchannel, and the specific steps are as follows: Step 1: Prepare a fluorescent polystyrene microsphere suspension, the specific method is as follows: 400 µL (1 wt%) of fluorescent polystyrene microspheres with diameters of 5 μm and 15 μm were taken into two test tubes to simulate the movement behavior of Chlorella and Haematococcus pluvialis, respectively. Deionized water was added to each of the two test tubes to dilute to 10 mL to prepare two single-sized particle suspensions, and then the two single-sized particle suspensions were mixed, and 100 µL of Tween 20 solution was added to the mixture, shaken evenly, and finally ultrasonicated to obtain a fluorescent polystyrene microsphere particle suspension.

[0024] Step 2: Prepare the microalgae cell mixture. The specific process is as follows: Step 2.1, cultivation of microalgae and collection of microalgae mixture: Cultivate common Chlorella and Haematococcus pluvialis in BG11 medium respectively, place in conical flasks, grow in a fully automatic intelligent light incubator, and receive a 12 / 12 hour light and dark cycle, and control the relevant temperature at 23-25°C. Shake the culture continuously every day to ensure the uniformity of nutrients and the dispersion of cells. At the beginning of 7-10 days, count the cells by using a hemocytometer and an inverted microscope; A phosphate buffer solution with a pH of 7.2-7.4 is taken and dripped into deionized water to prepare a PBS solution for use, wherein the volume ratio of deionized water to phosphate buffer solution is 10:1; Step 2.2, process the microalgae samples: take the cultured Haematococcus pluvialis and Chlorella, respectively, and coarsely filter the Haematococcus pluvialis and Chlorella with 20 µm and 10 µm filters, filter out the cell precipitate, and then filter out the oversized cells again with 18 µm and 6 µm precision filters, respectively. Then, put them into a high-speed desktop centrifuge at a speed of 6000-6500 rpm and a temperature of 20-22°C for 4-5 min, pour out the supernatant, take out the algae cells, put them into test tubes, add the prepared PBS solution to obtain two algae cell solutions, the PBS concentration range of each algae cell solution is 0.1-0.5M, finally, take out the same volume of Haematococcus pluvialis solution and Chlorella solution, mix them, add 100 µL of sorbitol solution to the mixed solution and shake it thoroughly to obtain a microalgae cell mixed solution, and the volume ratio of the mixed solution to the sorbitol solution is 200:1.

[0025] Among them, Chlorella vulgaris and Haematococcus pluvialis (growth period) were purchased from Shanghai Guangyu Biotechnology Co., Ltd. and cultured according to the requirements.

[0026] Step 3: Conduct velocity distribution simulation experiments on three types of microchannels to obtain simulation results. The specific method is as follows: In conventional spiral chips, inertial lift is the dominant mechanism, but its magnitude and direction depend on the uncertainty of the channel structure, making it difficult to quantitatively analyze the position of specific particles. However, with the coupling with the contraction-expansion channel, the vortex-induced lift is constantly directed toward the expansion area, greatly reducing the strong dependence of the normal spiral channel on the channel structure. The contraction-expansion structure can generate vortices, further strengthening the secondary flow, helping the particles to stack in an orderly manner and quickly reach equilibrium.

[0027] In CFD simulation, channel structure, flow rate, liquid medium density and viscosity are the main factors affecting particle separation. Figure 4 The flow field distribution on each circuit section is shown. Figure 5It can be seen that there is a considerable velocity gradient between the contraction and expansion zones, which accelerates the particle focusing process. During the particle migration process, smaller particles are not restricted by the chip structure and migrate and focus at the equilibrium position close to the inner or outer wall. Under the combined effect of vortex-induced lift and Dean drag, they remain stable before reaching the outlet. However, for larger particles, the vortex-induced lift in the contraction and expansion zones is relatively stable, and under the combined effect of vortex-induced lift and Dean drag, it further affects their equilibrium position.

[0028] In the simulation, the outlets of the channels are open, i.e. they can enter the atmosphere, which means that the output pressure is equal to atmospheric pressure, in keeping with the initial velocity conditions imposed at the inlet of a normal spiral channel. The conditions are different for the outlets with different inlet pressures. By using the inlet velocity as a variable for focusing the particles and correcting for the velocity, the expected conditions for the microchannel outlet are achieved. According to the Bernoulli relation, it can be expected that the pressure from the inlet to the outlet will decrease, eventually reaching atmospheric pressure at the outlet. Figure 6 It is the pressure drop that finally reaches the outlet atmospheric pressure after each cycle.

[0029] The characteristics of the designed spiral expansion and unilateral coupling geometry are enough to focus particles (cells) on a line. During the focusing process, particles are randomly injected into the inlet of the spiral expansion and unilateral coupling microchannel. The microchannel sorts the particles on a line (the center line of the channel) through each round of circulation. The spiral expansion and unilateral coupling geometry structure completes the cell focusing process well. The focusing effect of 5 µm particles was studied using the spiral expansion and unilateral coupling microchannel. The spiral expansion and unilateral coupling microchannel with a speed of 1m / s has good particle focusing performance. The results of particle focusing by the spiral expansion and unilateral coupling geometry are shown in Figure 2. Figure 7 As shown in (a), the focusing effect of 5 µm particles at a flow rate of 150 µL / min in the comparative verification experiment is not much different. Figure 7 (b) shown.

[0030] Step 4, using fluorescent polystyrene microsphere particle suspension to conduct preliminary experiments on the inertial migration behavior of particles in three different microchannels, and obtain preliminary experimental results; Before separating the actual algae samples, we first used fluorescent polystyrene particles of similar size to characterize the inertial migration behavior of particles in a spiral converging and expanding single-sided coupled flow channel microchannel. The specific process is as follows: The microfluidic device was mounted on a bracket, and two fluorescent polystyrene microsphere particle suspensions were injected into the channel using a syringe pump, wherein the flow rate during injection into the channel was 50 µL / min, 100 µL / min, 150 µL / min, 200 µL / min, 300 µL / min, or 400 µL / min, and a constant flow rate of 10-400 μL / min was maintained. The entire device was then placed on the stage of an inverted microscope, and a high-speed camera was used to monitor and record the flow of the sample in the microchannel; The inertial separation behavior of microspheres with different diameters was analyzed using an optimized microchannel design. These polystyrene microspheres were labeled with fluorescent dyes, allowing us to clearly see the tracking trajectory. The particles that were finally focused into a single trajectory obey the empirical relationship, expressed as This shows that the polystyrene microspheres we used can fully achieve inertial focusing and reach a steady state. We injected two types of fluorescent microspheres into the channel at different flow rates to explore the optimal flux.

[0031] In the curved channel near the entrance, particles of different sizes showed similar behavior. They were evenly distributed, had no focusing effect, and completely occupied the entire cross-section of the channel. In the second cycle of the channel, the particles began to show a tendency to concentrate at their own equilibrium position. 5μm fluorescent polystyrene microspheres were used to simulate the behavior of Chlorella in the microchannel, and the focusing effect of the particles was compared at flow rates of 50µL / min, 100µL / min, 150µL / min, 200 µL / min, 300 µL / min, and 400 µL / min; Figure 8 As shown in the figure, the particle focusing effect is best at flow rates of 50 µL / min, 100 µL / min, and 150 µL / min, achieving a single-line focusing effect. The particle focusing effect is second best at flow rates of 200 µL / min and 300 µL / min, achieving a double-line focusing effect. There is no focusing effect at a flow rate of 400 µL / min, and most of the particles are scattered at the second outlet, and a small number of particles flow out from the first outlet. 15 µm fluorescent polystyrene particles were used to simulate the behavior of Haematococcus pluvialis in the microchannel. Analogously to the focusing analysis of 5 µm fluorescent polystyrene particles, the flow rate of 15 µm fluorescent polystyrene particles was increased and compared. The flow rate was controlled at 100-400 µL / min, with a gradient of 100, and the focusing effects of the particles at flow rates of 100 µL / min, 200 µL / min, 300 µL / min and 400 µL / min were compared, as shown in Figure 1. Fig. 9As shown in the figure, the particle focusing effect of 15 µm fluorescent polystyrene particles is very ideal at flow rates of 100 µL / min, 200 µL / min, 300 µL / min and 400 µL / min, forming a single-line focus. At a flow rate of 100 µL / min, 15 µm fluorescent polystyrene particles all flow out from the first outlet, and at flow rates of 200 µL / min, 300 µL / min and 400 µL / min, 15 µm fluorescent polystyrene particles all flow out from the second outlet.

[0032] By analyzing the focusing conditions of the two fluorescent polystyrene particles, the optimal flow rate for sorting the two mixed particles was 100-200 µL / min.

[0033] Step 5. Based on the preliminary experimental results, three different spiral and contraction-expansion coupled microchannels were used to experimentally study the inertial separation behaviors of Chlorella and Haematococcus pluvialis, and the performance of three different microchannel chips for flexible microalgae sorting was evaluated to obtain the optimal separation parameters and complete the sorting of flexible microalgae.

[0034] The focusing analysis of 5µm and 15µm fluorescent polystyrene particles provides a good reference for the focusing of the two microalgae. Unlike the relatively consistent size of commercial fluorescent polystyrene microspheres, the size of microalgae cells is polydisperse. Due to differences in growth stage and culture state, the size of Haematococcus pluvialis can be dispersed between 10 µm and 25 µm. Under the same conditions, the two microalgae cells were respectively introduced into three different microchannels using a syringe pump, and the flow rate when injected into the channel was 100 to 400µL / min.

[0035] In this embodiment, the specific method of using the spiral contraction and expansion single-side coupling microchannel chip for flexible microalgae sorting is: Based on the focusing of two fluorescent polystyrene microspheres and two types of microalgae at different flow rates, the mixed microalgae samples were sorted. By analyzing the focusing effect of the two cells at different flow rates, it is estimated that 200 µL / min has a better separation effect. Fig.10 This is the separation effect diagram of mixed microalgae at 200 µL / min, where Fig.10 (a) is the particle trajectory diagram of the mixed microalgae focusing at the outlet of the spiral and contraction-expansion channel unilateral coupling sorting microchannel. It can be clearly seen that Chlorella and Haematococcus pluvialis flow out from the first outlet and the second outlet respectively. Fig.10 (b) is a grayscale image of the particle trajectory of the mixed microalgae at the exit of the spiral and contraction-expansion channel unilateral coupling sorting microchannel, which can be used to quantitatively study the Fig.10 Grayscale value of the intersection line MM at (a). The focal position of Chlorella vulgaris is 96 µm from the upper boundary of the flow channel, and the focal position of Haematococcus pluvialis is 239 µm from the upper boundary of the flow channel.

[0036] In addition to visual confirmation using a high-speed camera, the performance of the spiral and contraction-expansion channel unilateral coupling sorting chip microchannel can also be evaluated in terms of cell purity and recultivability. Among them, the separation performance is mainly characterized by purity, and the specific formula for calculating purity EP is: EP = N a (outlet i ) / N total (outlet i ) (1) In the formula, N a (outlet i ) is the number of target cell population a in outlet i, N total (outlet i ) is the total number of cells extracted from this outlet.

[0037] Under the optimal operating conditions, the flow rate was 200 µL / min, and samples were collected from outlets 1 and 2 for quantitative analysis. By measuring the concentration of Chlorella cells and Haematococcus pluvialis cells in the collected liquid from each outlet, the total number of cells contained therein can be calculated, thereby calculating the purity of the microalgae cells at each outlet. Fig.11 As shown in the figure, the purity of Chlorella produced from the first outlet is 100%, and the purity of Haematococcus pluvialis produced from the second outlet is 76.5%. The reasons for the low purity of the second outlet are: first, the focusing law of Chlorella will cause some cells to focus at the second outlet when it is 200 µL / min; second, the cell division speed of Chlorella is fast and the relatively high cell concentration leads to intensified collisions between particles, causing some particles to move irregularly.

[0038] Due to the one-sided coupling of the spiral and contraction-expansion channels, at high flow rates, microalgae cells will generate large pressure and high shear stress when passing through the contraction chamber, which may cause damage to the cells. Therefore, recultivation capacity is another key performance indicator. The recultivation capacity can be evaluated by re-inoculating the isolated algae cells into new culture medium. Observe the algae cells after 7 days of separation and re-cultivation. Fig.12 The results show that both cell types grow well and can continue to grow. The Haematococcus pluvialis is very active. In the pictures, it can be observed that there are afterimages in the movement trajectory of the Haematococcus pluvialis, and there is no sign of any cell damage.

[0039] Since the separation method based on three coupled microchannels is only effective when the algae cells are separated, we cultured the separated microalgae in test tubes for subsequent use. 1 mL of the collected algae cells was added to each test tube, and 1 mL of BG 11 medium was added for nutrient supplementation of Chlorella and Haematococcus pluvialis. The microalgae cell samples from the three outlets were cultured at 23 °C for 7 days using the initial culture method, and shaken every 1 h to prevent sedimentation.

[0040] Example 2 The method for flexible microalgae sorting based on spiral and contraction-expansion coupled microchannel uses the spiral and contraction-expansion coupled microchannel, and the specific steps are as follows: The specific methods and parameters of steps 1 to 4 are the same as those of Example 1. The difference from Example 1 is that in step 4, the separation of Chlorella and Haematococcus pluvialis is analyzed by using a spiral contraction-expansion double-sided coupling flow channel. By analyzing the focusing effects of the two cells at different flow rates, the flow rate with the best separation effect is obtained. The specific method is: By analogy with the spiral contraction-expansion single-side coupling flow channel, the separation of Chlorella vulgaris and Haematococcus pluvialis was analyzed, and the flow rate was preferably 150 µL / min and 200 µL / min, such as Fig.13 As shown in the figure, at the two flow rates, the microalgae mixture flows in disorderly from the inlet, and then after passing through the first, second, and third ring microfluidics, the two microalgae are gradually focused and separated, and finally Chlorella flows out from the first outlet and Haematococcus pluvialis flows out from the second outlet. By comparing the focusing and sorting states at the two flow rates, the focusing width of Chlorella at a flow rate of 150 µL / min is wider than that at a flow rate of 200 µL / min, and the focusing width of Haematococcus pluvialis at a flow rate of 150 µL / min is narrower than that at a flow rate of 200 µL / min.

[0041] The two outlet solutions of the microfluidic channel were collected and the cells in the outlet solution were counted using a blood cell counting chamber and an inverted microscope. Fig.14 As shown in the figure, the solution composition at outlet 1 is mainly Chlorella and a very small part of Haematococcus pluvialis, and the solution composition at outlet 2 is mainly Haematococcus pluvialis and part of Chlorella. It can be seen from the collection of the outlet solution that the collection concentration at outlet 2 is higher. Due to the bilateral coupling of the spiral and contraction-expansion channels, Chlorella is greatly affected by the vortex-induced lift, resulting in some unstabilized focusing of Chlorella at outlet 2, while Haematococcus pluvialis is impacted by the incompletely stably focused Chlorella and remains at the first outlet.

[0042] Under the optimal operating conditions, the flow rate was 200 µL / min, and samples were collected from outlets 1 and 2 for quantitative analysis. By measuring the concentration of Chlorella cells and Haematococcus pluvialis cells in the collected solution from each outlet, the total number of cells contained therein can be calculated, thereby calculating the purity of the microalgae cells at each outlet, such as Fig.15 As shown, the results showed that the purity of Chlorella produced from the first outlet was 92.2%, and the purity of Haematococcus pluvialis produced from the second outlet was 61.5%.

[0043] Similarly, the double-side coupling of the spiral channel and the contraction-expansion channel will generate high pressure and high shear stress when passing through the contraction chamber under high flow rate, which may cause damage to the cells. Therefore, the re-cultivation ability is evaluated by re-inoculating the separated algae cells into new culture medium. Fig.16 The results show that both cell types grow well and can continue to grow. The Haematococcus pluvialis is very active. In the pictures, it can be observed that there are afterimages in the movement trajectory of the Haematococcus pluvialis, and there is no sign of any cell damage.

[0044] Example 3 The method for flexible microalgae sorting based on spiral and contraction-expansion coupled microchannel uses the spiral and contraction-expansion coupled microchannel, and the specific steps are as follows: The specific methods and parameters of steps 1 to 4 are the same as those of Example 1. The difference from Example 1 is that in step 4, the separation of Chlorella and Haematococcus pluvialis is analyzed by using a spiral contraction-expansion staggered coupling flow channel. By analyzing the focusing effect of the two cells at different flow rates, the flow rate with the best separation effect is obtained. The specific method is: Similarly, by analogy with the spiral contraction-expansion single-side coupling flow channel, the separation of Chlorella vulgaris and Haematococcus pluvialis was analyzed, and the flow rates were preferably 150 µL / min and 200 µL / min. Fig.17 As shown in the figure, at the two flow rates, the microalgae mixture flows in disorderly from the inlet, and then after passing through the first, second, and third rings of the microfluidic channel, the two microalgae are gradually focused and separated, and finally Chlorella flows out from the first outlet and Haematococcus pluvialis flows out from the second outlet. By comparing the focusing and sorting states at the two flow rates, the focusing width of Chlorella at the two flow rates is similar, and the focusing position at a flow rate of 150 µL / min is closer to the outer wall of the flow channel than the focusing position at a flow rate of 200 µL / min. At the two flow rates, the focusing width of Haematococcus pluvialis at a flow rate of 150 µL / min is narrower than the focusing width at a flow rate of 200 µL / min.

[0045] Similarly, the solutions at the two outlets of the microfluidic channel were collected and the cells in the outlet solutions were counted using a blood cell counting chamber and an inverted microscope. Fig.18As shown, the composition of the solution at outlet 1 is mainly Chlorella and a small part of Haematococcus pluvialis, and the composition of the solution at outlet 2 is mainly Haematococcus pluvialis and part of Chlorella. It can be seen from the collection of the outlet solution that the collection concentration at outlet 2 is higher. Due to the staggered coupling of the spiral and contraction-expansion channels, Chlorella is affected by the vortex-induced lift and is greatly affected by it. The direction of the vortex-induced lift on Chlorella changes too rapidly due to the staggered coupling. In the end, Chlorella will still have some unstable focusing at outlet 2, while Haematococcus pluvialis will be impacted by the incompletely stable focusing Chlorella and will remain at the first outlet.

[0046] Under the optimal operating conditions, the flow rate was 200 µL / min, and samples were collected from outlets 1 and 2 for quantitative analysis. By measuring the concentration of Chlorella cells and Haematococcus pluvialis cells in the collected liquid from each outlet, the total number of cells contained therein can be calculated, thereby calculating the purity of the microalgae cells at each outlet. Fig.19 As shown, the results showed that the purity of Chlorella produced from the first outlet was 90.1%, and the purity of Haematococcus pluvialis produced from the second outlet was 51.2%.

[0047] Similarly, the staggered coupling of spiral and contraction-expansion channels, under high flow rate, will generate high pressure and high shear stress when repeatedly passing through the contraction neck and entering the expansion cavity, which may cause damage to the cells. Therefore, the re-cultivation ability was evaluated by re-inoculating the separated algae cells into new culture medium. Fig. 20 The results show that both cells grow well and can continue to grow. The Haematococcus pluvialis is very active. In the picture, it can be observed that the movement trajectory of the Haematococcus pluvialis has residual images, and there is no sign of any cell damage. In addition, the separation itself produces pure single-cell culture, which can better cultivate single Chlorella cells and Haematococcus pluvialis with higher purity than ordinary inertial spiral chips. In this embodiment, in order to facilitate better focusing, algae samples with low concentrations were used. If natural water samples with much lower algae cell concentrations are used, it is possible to obtain higher purity. In addition, dilution or multiple separations of the initial sample can also be used as a strategy to improve purity.

[0048] Example 4 The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel, the specific steps are as follows: Step 1, preparing a fluorescent polystyrene microsphere particle suspension; Step 2, preparing a microalgae cell mixture; Step 3, using fluorescent polystyrene microsphere particle suspension to simulate the inertial migration behavior of particles in three different microchannels, and obtaining simulation results; Step 4: Based on the simulation results, three different spiral and contraction-expansion coupled microchannels were used to experimentally study the inertial separation behaviors of Chlorella and Haematococcus pluvialis, and the performance of three different microchannel chips for flexible microalgae sorting was evaluated to obtain the optimal separation parameters and complete the sorting of flexible microalgae.

[0049] Example 5 The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel, the specific steps are as follows: Step 1. Prepare a fluorescent polystyrene microsphere particle suspension. The specific method is as follows: take fluorescent polystyrene microspheres with diameters of 5 μm and 15 μm respectively, add deionized water to dilute, and prepare two single-sized particle suspensions, wherein the volume ratio of each fluorescent polystyrene microsphere to deionized water is 1:24, and then mix the two single-sized particle suspensions at a volume ratio of 1:1, add Tween 20 solution to the mixed solution, the volume ratio of the Tween 20 solution to the mixed solution is 1:200, oscillate evenly, and finally obtain a fluorescent polystyrene microsphere particle suspension by ultrasonic treatment.

[0050] Step 2, preparing a microalgae cell mixture; Step 3, using fluorescent polystyrene microsphere particle suspension to simulate the inertial migration behavior of particles in three different microchannels, and obtaining simulation results; Step 4: Based on the simulation results, three different spiral and contraction-expansion coupled microchannels were used to experimentally study the inertial separation behaviors of Chlorella and Haematococcus pluvialis, and the performance of three different microchannel chips for flexible microalgae sorting was evaluated to obtain the optimal separation parameters and complete the sorting of flexible microalgae.

[0051] Example 6 The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel, the specific steps are as follows: Step 1, preparing a fluorescent polystyrene microsphere particle suspension; Step 2: Prepare the microalgae cell mixture. The specific process is as follows: Step 2.1, cultivation of microalgae and collection of microalgae mixture: Cultivate common Chlorella and Haematococcus pluvialis in BG11 medium respectively, place in conical flasks, grow the culture in a fully automatic intelligent light incubator, and receive a 12 / 12 hour light and dark cycle, the relevant temperature is controlled at 23°C, and the culture is continuously shaken every day to ensure the uniformity of nutrients and the dispersion of cells. At the beginning of 7-10 days, the cells are counted by using a hemocytometer and an inverted microscope; A phosphate buffer solution with a pH of 7.2-7.4 is taken and dripped into deionized water to prepare a PBS solution for use, wherein the volume ratio of deionized water to phosphate buffer solution is 10:1; Step 2.2, process the microalgae samples: take the cultured Haematococcus pluvialis and Chlorella, respectively, and coarsely filter the Haematococcus pluvialis and Chlorella with 20 µm and 10 µm filters, filter out the cell precipitate, and then filter out the oversized cells again with 18 µm and 6 µm precision filters, respectively. Then, put them into a high-speed desktop centrifuge at 6000 rpm and centrifuge at 20°C for 4 min, pour out the supernatant, take out the algae cells, put them into test tubes, add the prepared PBS solution to obtain two algae cell solutions, the PBS concentration range of each algae cell solution is 0.1~0.5M, finally, take out the same volume of Haematococcus pluvialis solution and Chlorella solution, mix them, add 100 µL of sorbitol solution to the mixed solution and shake it thoroughly to obtain a microalgae cell mixed solution, and the volume ratio of the mixed solution to the sorbitol solution is 200:1.

[0052] Among them, Chlorella vulgaris and Haematococcus pluvialis (growth period) were purchased from Shanghai Guangyu Biotechnology Co., Ltd. and cultured according to the requirements.

[0053] Step 3, using fluorescent polystyrene microsphere particle suspension to simulate the inertial migration behavior of particles in three different microchannels, and obtaining simulation results; Step 4: Based on the simulation results, three different spiral and contraction-expansion coupled microchannels were used to experimentally study the inertial separation behaviors of Chlorella and Haematococcus pluvialis, and the performance of three different microchannel chips for flexible microalgae sorting was evaluated to obtain the optimal separation parameters and complete the sorting of flexible microalgae.

Claims

1. Spiral and contraction-expansion coupled microchannel, characterized in that, It is composed of a spiral microchannel and a contraction-expansion channel coupled in three different forms: single-side coupling, double-side coupling or staggered coupling. The spiral and contraction-expansion coupled microchannel has a sample inlet and two outlets. The initial radius of the spiral and contraction-expansion coupled microchannel is 3-5 mm. The cross-section of the spiral microchannel is rectangular, the contraction cavity and the expansion cavity in the spiral and contraction-expansion coupling microchannel are arranged at an equal angle of 5°, the cross-sectional size of the contraction cavity is 50ⅹ50µm, and the cross-sectional size of the expansion cavity is 200ⅹ50µm.

2. A method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannels, characterized in that: Using the spiral and contraction-expansion coupling microchannel as claimed in claim 1, the specific steps are as follows: Step 1, preparing a fluorescent polystyrene microsphere particle suspension; Step 2, preparing a microalgae cell mixture; Step 3, conduct velocity distribution simulation experiments on three types of microchannels to obtain simulation results; Step 4: Based on the simulation results, a fluorescent polystyrene microsphere particle suspension is used to conduct preliminary experiments on the inertial migration behavior of particles in three different microchannels to obtain preliminary experimental results; Step 5. Based on the preliminary experimental results, three different spiral and contraction-expansion coupled microchannels were used to analyze and compare the inertial separation behaviors of Chlorella and Haematococcus pluvialis, and the performance of three different microchannel chips for flexible microalgae sorting was evaluated to obtain the optimal separation parameters and complete the sorting of flexible microalgae.

3. The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel according to claim 2, characterized in that: The specific method of step 1 is: taking fluorescent polystyrene microspheres with diameters of 5 μm and 15 μm respectively, adding deionized water to dilute, and preparing two single-sized particle suspensions, wherein the volume ratio of each fluorescent polystyrene microsphere to deionized water is 1:24, and then mixing the two single-sized particle suspensions at a volume ratio of 1:1, adding Tween 20 solution to the mixed solution, wherein the volume ratio of the Tween 20 solution to the mixed solution is 1:200, oscillating evenly, and finally obtaining a fluorescent polystyrene microsphere particle suspension by ultrasonic treatment.

4. The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel according to claim 2, characterized in that: The specific process of step 2 is: Step 2.1, cultivation of microalgae and collection of microalgae mixture: Cultivate common Chlorella and Haematococcus pluvialis in BG11 medium respectively, place in conical flasks, grow the cultures in a fully automatic intelligent light incubator, and receive a 12 / 12 hour light and dark cycle, and control the relevant temperature at 23-25°C. Shake the cultures continuously every day to ensure uniform nutrients and dispersion of cells; A phosphate buffer solution with a pH of 7.2-7.4 is taken and dripped into deionized water to prepare a PBS solution for standby use, wherein the volume ratio of the deionized water to the phosphate buffer solution is 10:1; Step 2.2, treating the microalgae samples: taking the cultured Haematococcus pluvialis and Chlorella, respectively, coarsely filtering the Haematococcus pluvialis and Chlorella with 20 µm and 10 µm filters, filtering out the cell precipitate, and then filtering out the oversized cells again with 18 µm and 6 µm precision filters, respectively, and then placing them in a high-speed desktop centrifuge at a speed of 6000-6500 rpm and a temperature of 20-22°C for 4-5 min, pouring out the supernatant, taking out the algae cells, and adding the prepared PBS solution to obtain two algae cell solutions, each of which has a PBS concentration range of 0.1-0.5M. Finally, taking out the same volume of Haematococcus pluvialis solution and Chlorella solution, respectively, mixing them, adding sorbitol solution to the mixed solution and shaking it thoroughly to obtain a microalgae cell mixed solution, and the volume ratio of the mixed solution to the sorbitol solution is 200:

1.

5. The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel according to claim 2, characterized in that: The specific process of step 4 is as follows: The microfluidic device was mounted on a bracket, and two fluorescent polystyrene microsphere particle suspensions were injected into the channel using a syringe pump, wherein the flow rate during injection into the channel was 50 µL / min, 100 µL / min, 150 µL / min, 200 µL / min, 300 µL / min, or 400 µL / min, and a constant flow rate of 10-400 μL / min was maintained. The entire device was then placed on the stage of an inverted microscope, and a high-speed camera was used to monitor and record the flow of the sample in the microchannel; The 5 μm fluorescent polystyrene microspheres are used to simulate the behavior of Chlorella in the microchannel, and the focusing effects of the particles are compared at flow rates of 50 μL / min, 100 μL / min, 150 μL / min, 200 μL / min, 300 μL / min and 400 μL / min; The 15 µm fluorescent polystyrene particles are used to simulate the behavior of Haematococcus pluvialis in the microchannel, and the flow rate of the 15 µm fluorescent polystyrene particles is increased, and the flow rate is controlled at 100-400 µL / min, with 100 as a gradient, and the focusing effect of the particles at the flow rates of 100 µL / min, 200 µL / min, 300 µL / min and 400 µL / min is compared respectively; By analyzing the focusing conditions of the two fluorescent polystyrene particles, the optimal flow rate for sorting the two mixed particles was 100-200 µL / min.

6. The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel according to claim 2, characterized in that: In step 5, a syringe pump is used to respectively introduce the two types of microalgae cells into three different microchannels, wherein the flow rate when injecting into the channel is 100-400 μL / min.

7. The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel according to claim 6, characterized in that: The specific method of using the spiral contraction and expansion single-side coupling microchannel chip to perform flexible microalgae sorting in step 5 is: The separation of Chlorella vulgaris and Haematococcus pluvialis was analyzed using a spiral contraction-expansion single-sided coupling flow channel. By analyzing the focusing effects of the two cells at different flow rates, it was found that the flow rate with the best separation effect was 200 µL / min.

8. The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel according to claim 6, characterized in that: The specific method of the method for flexible microalgae sorting using a spiral contraction and expansion double-sided coupling microchannel chip in step 5 is: The separation of Chlorella vulgaris and Haematococcus pluvialis was analyzed using a spiral contraction-expansion double-sided coupling flow channel. By analyzing the focusing effects of the two cells at different flow rates, it was found that the flow rate with the best separation effect was 200 µL / min.

9. The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel according to claim 6, characterized in that: The specific method of using the spiral contraction and expansion staggered coupling microchannel chip to perform flexible microalgae sorting in step 5 is: The separation of Chlorella vulgaris and Haematococcus pluvialis was analyzed using a spiral contraction-expansion staggered coupling flow channel. By analyzing the focusing effects of the two cells at different flow rates, it was found that the flow rate with the best separation effect was 200 µL / min.

10. The method for flexible microalgae sorting based on spiral and contraction-expansion coupling microchannel according to claim 6, characterized in that: When evaluating the performance of flexible microalgae sorting using three different microchannel chips in step 5, in addition to visual confirmation using a high-speed camera, the indicators can also be evaluated from aspects such as cell purity and recultivability; Among them, the specific formula for calculating purity EP is: EP = N a (outlet i ) / N total (outlet i ) （1） In the formula, N a (outlet i ) is the number of target cell population a in outlet i, N total (outlet i ) is the total number of cells extracted from this outlet.

Images